α-subunit of the Mac-1 leukocyte adhesion receptor

ABSTRACT

The present invention relates to Mac-1 alpha-subunit which is involved in the process through which cells recognize and migrate to sites of inflammation as well as attach to cellular substrates during inflammation. The invention is directed toward such molecules, the functional derivatives of such molecules, screening assays for identifying such molecules and therapeutic and diagnostic uses for such molecules.

CROSS-REFEERNCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application No.08/077,098, filed Jun. 16, 1993, (abandoned); which is a continuation ofSer. No. 07/942,056, filed Sep. 8, 1992, (abandoned); which is acontinuation of Ser. No. 07/321,239, filed Mar. 9, 1989, (abandoned);which is a continuation-in-part of Ser. No. 07/235,353, filed Aug. 23,1988, (abandoned).

FIELD OF THE INVENTION

The present invention relates to the leukocyte adhesion receptor Mac-1.The invention further pertains to the cloning of DNA sequences whichencode the alpha-subunit of this molecule. This invention was made inpart with government support. The government has certain rights in thisinvention.

DESCRIPTION OF THE RELATED ART

The immune system is responsible for protecting an animal from foreigninvaders, such as bacteria, viruses, etc. An excellent review of thedefense system is provided by Eisen, H. W. (In: Microbiology, 3rd Ed.,Harper & Row, Philadelphia, Pa. (1980), pp. 290-295 and 381-418). Theability of the immune system to protect an animal against foreigninvaders depends, in large measure, on the presence and function ofblood cells known an leukocytes. The ability of leukocytes to providesuch protection has been found to require that these cells adhere tocellular and extracellular substrates.

For example, leukocytes must be able to attach to endothelial cells sothat they can migrate from circulation to sites of ongoing inflammation.Furthermore, they must attach to antigen-presenting cells so that anormal immune response can occur. They must also be able to attach toappropriate target cells so that the lysis of virally-infected (ortumor) cells can occur. Furthermore, leukocytes must be able to attachto various activated proteins (such as iC3b--the activated form of thethird component of complement) so that they may efficiently phagocytoseand clear microbial and cellular debris. Thus, leukocyte adhesion is arequisite of a normally functioning host defense system. The inhibitionof this defense system is desirable in cases such as transplantation,because the host "sees" the transplanted tissue as foreign and initiatesan immune response to that tissue. Leukocyte adhesion is, therefore,also involved in the rejection of transplanted tissue and organs. Thus,an understanding of leukocyte adhesion may enable one to either augmentan animal's ability to fight infection or suppress an animal's capacityto reject transplanted tissue.

Recently, leukocyte surface molecules involved in mediating leukocyteadhesion were identified using hybridoma technology. Briefly, monoclonalantibodies directed against human T-cells (Davignon, D., et al., Proc.Natl. Acad. Sci. USA 78:4535-4539 (1981)) and mouse spleen cells(Springer, T., et al., Eur. J. Immunol. 9:301-306 (1979)) wereidentified which bound to leukocyte surfaces and inhibited theattachment-related functions described above (Springer, T., et al., Fed.Proc. 44:2660-2663 (1985)). The molecules which were recognized by theseantibodies comprise a set of leukocyte adhesion receptors known as the"Lymphocyte Function-Associated Antigen-1 family" (or the "LFA-1family") of adhesion receptor molecules.

The LFA-1 family of adhesion receptor molecules contains three highlyrelated cell surface glycoproteins. These glycoproteins have been foundto mediate cell-cell interactions in inflammation. The glycoproteinshave been designated "LFA-1" (lymphocyte function-associated antigen-1),"Mac-1" and "p150,95." Whereas LFA-1 is found on the surfaces of mostleukocytes (Springer, T. A., et al., Immunol. Rev. 68.:111-135 (1982)),Mac-1 and p150,95 are found primarily on macrophages, granulocytes andother large granular lymphocytes (Springer, T. A., et al., Immunol. Rev.68:111-135 (1982); Keizer, G., et al., Eur. J. Immunol. 15:1142-1147(1985)).

The LFA-1 glycoprotein family is composed of heterodimers, eachcontaining an alpha-subunit which is non-covalently associated with abeta-subunit. The alpha-subunits of the family have been found to differfrom one another and are designated CD11a, CD11b, and CD11c,respectively. The glycosylated alpha-subunits have approximate molecularweights of 180, 170, and 150 kd, respectively. In contrast, thebeta-subunit of the LFA-1 family of adhesion receptors has been found tobe identical, and to have a molecular weight of 95 kd (Sanchez-Madrid,F., et al., J. Exper. Med. 158:1785-1803 (1983); Keizer, G. D., et al.,Eur. J. Immunol. 15:1142-1147 (1985); Springer, T., Fed. Proc.44:2660-2663 (1985); Sanchez-Madrid, F., et al., J. Exper. Med.158:586-602 (1983)).

Although the alpha-subunits of the glycoproteins do not exhibit theextensive homology shared by the beta-subunits, close analysis of thealpha-subunits of the glycoproteins has revealed that there aresubstantial similarities between them. Reviews of the similaritiesbetween the alpha and beta-subunits of the adhesion moleculeglycoprotein family are provided by Sanchez-Madrid, F., et al. (J.Exper. Med. 158:586-602 (1983); J. Exper. Med. 158:1785-1803 (1983);Miller, L. J., et al., J. Immunol. 138:2381-2383 (1987)).

The importance of the LFA-1 family of receptors was initially recognizedin studies which showed the ability of monoclonal antibodies (which werecapable of binding to either the specific alpha-subunits, or the commonbeta-subunit) to inhibit adhesion-dependent leukocyte functions(Sanchez-Madrid, F., et al., Proc. Natl. Acad. Sci. USA 79:7489-7493(1982); Beller, D. I., et al., J. Exper. Med. 156:1000-1009 (1982)).

Recently, a group of individuals has been identified who are unable toexpress normal amounts of any member of the Mac-1 adhesion proteinfamily on their leukocyte cell surfaces. This disease has been termed"Leukocyte Adhesion Deficiency" or "LAD" and is characterized by chronicand recurring infections, as well as other clinical symptoms (Anderson,D. C., et al., Fed. Proc. 44:2671-2677 (1985); Anderson, D. C., et al.,J. Infect. Dis. 152:668-689 (1985)). Leukocytes from LAD patientsdisplay in vitro defects which were similar to those observed whenleukocytes of normal individuals were antagonized by antibody specificfor members of the LFA-1 family. LAD patients were found to be unable tomount a normal immune response. This failure was found to be due to aninability of the leukocytes of LAD patients to adhere to cellular andextracellular substrates (Anderson, D. C., et al., Fed. Proc.44:2671-2677 (1985); Anderson, D. C., et al., J. Infect. Dis.152:668-689 (1985)). These studies show that inflammatory reactions aremitigated when leukocytes are unable to adhere in a normal fashion dueto the lack of functional adhesion molecules on their cell surface.

Thus, in summary, the ability of leukocytes to maintain the health andviability of an animal requires that they be capable of adhering toother cells (such as endothelial cells) and proteins (such as iC3b).This adherence has been found to require contacts which involve specificreceptor molecules present on the leukocyte surface of the leukocytes.These cell surface receptor molecules have been found to be highlyrelated to one another. Humans whose leukocytes lack these cell surfacereceptor molecules exhibit chronic and recurring infections, as well asother clinical symptoms.

Since leukocyte adhesion is involved in the process through whichforeign tissue is identified and rejected, an understanding of thisprocess is of significant value in the fields of organ transplantation,tissue grafts, allergy and oncology.

SUMMARY OF THE INVENTION

The present invention relates to leukocyte cell surface adhesionreceptor molecules, and in particular, to the cloning and expression ofthe alpha-subunit of the Mac-1 receptor molecule through the use ofrecombinant DNA technology. The invention pertains to the adhesionmolecule itself, to functional fragments of the molecule, to nucleicacid (i.e., DNA, and especially cDNA) capable of encoding these receptormolecules, and to plasmids which contain such nucleic acid sequences.The present invention additionally encompasses methods for producing thereceptor molecules which employ recombinant DNA technology.

In detail, the invention pertains to Mac-1 alpha-subunit, or afunctional derivative thereof, substantially free of naturalcontaminants.

The invention further pertains to the above Mac-1 alpha-subunit or thefunctional derivative thereof, which is additionally capable of bindingto a molecule present on the surface of a cell.

The invention also includes the above Mac-1 alpha-subunit molecule,wherein the molecule contains at least one polypeptide selected from thegroup consisting of:

    ______________________________________                                        a.         A--N--Q--R--G--S--L;                                               b.         M--E--Q--L--K--K--S;                                               c.         T--D--G--E--K--F--G;                                               d.         G--V--F--L--Y--T--S;                                               e.         V--D--V--D--S--S--N--G--S--T;                                      f.         D--V--N--G--D--K--L--T--D--V--A;                                   g.         D--L--T--M--D--G--L--V--D--L;                                      h.         Y--I--L--T--S--H--N;                                               i.         C--Q--D--D--L--S--I;                                               j.         T--I--Q--N--Q--L--R;                                               k.         V--Q--S--L--V--L--G;                                               l.         Y--Q--H--I--G--L--V;                                               m.         L--F--T--A--L--F--P; and                                           n.         F--S--L--V--G--T--P;                                               ______________________________________                                    

The invention also includes a recombinant DNA molecule capable ofexpressing either the Mac-1 alpha-subunit or a functional derivativethereof.

The invention also provides a method for recovering Mac-1 alpha-subunitin substantially pure form which comprises the steps:

(a) solubilizing Mac-1 alpha-subunit from the membranes of cellsexpressing Mac-1 alpha-subunit, to form a solubilized Mac-1alpha-subunit preparation,

(b) introducing the solubilized Mac-1 alpha-subunit preparation to anaffinity matrix, the matrix containing immobilized antibody capable ofbinding to Mac-1 alpha-subunit,

(c) permitting the Mac-1 alpha-subunit to bind to the antibody of theaffinity matrix,

(d) removing from the matrix any compound incapable of binding to theantibody and

(e) recovering the Mac-1 alpha-subunit in substantially pure form byeluting the Mac-1 alpha-subunit from the matrix.

The invention also provides a method for treating inflammation resultingfrom a response of the non-specific defense system (such as asthma;adult respiratory distress syndrome; multiple organ injury syndromesecondary to septicemia; multiple organ injury syndrome secondary totrauma; reperfusion injury of tissue; acute glomerulonephritis; reactivearthritis; dermatosis with acute inflammatory components; a centralnervous system inflammatory disorder; thermal injury; hemodialysis;leukapheresis; ulcerative colitis; Crohn's disease; necrotizingenterocolitis; granulocyte transfusion associated syndrome; andcytokine-induced toxicity) in a mammalian subject which comprisesproviding to a subject in need of such treatment an anti-inflammatoryagent, in an amount sufficient to suppress said inflammation; whereinsaid anti-inflammatory agent is selected from the group consisting of:the Mac-1 alpha-subunit; and a functional derivative of the Mac-1alpha-subunit.

The invention also pertains to the above method which additionallycomprises the co-administration of an agent selected from the groupconsisting of: the Mac-1 beta-subunit, and a functional derivative ofthe Mac-1 beta-subunit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the restriction map of Mac-1 alpha-subunit cDNA clones andsequencing strategy. The thick line represents the coding region of thecDNA. The polyadenylation signals are shown as black boxes. The regionsdeleted in λM90 is indicated as a dotted line. The indicated restrictionsites are Bal I (B), Bam H1 (Ba), Bgl II (Bg), Eco RI (E), Eco RV (Ev),Hinc II (Hc), Hind III (H), Sma I (S), and Pst I (P).

FIGS. 2A-2H show the nucleotide and cDNA-derived amino acid sequence ofthe alpha-subunit of Mac-1. The putative N-glycosylation sites areunderlined. The signal sequence and the putative transmembrane regionare indicated by a dotted line. The termination condon is labeled withan asterisk. Boxes in the 3' untranslated region show thepolyadenylation signal sequences. Arrows mark the boundaries of apotential intron spliced out of some of the cDNAs.

FIG. 3 shows Mac-1 α homologous repeats. Common residues are boxed. Thethree repeats containing putative divalent cation-binding sites (V-VII)are aligned with additional N-terminal related sequences lacking theputative Ca⁺⁺ or MG⁺⁺ -binding sites (I-IV). based on the frequency ofeach residue in the seven repeats of the α-subunits of Mac-1 and ofother integrins (p150,95, the frronectin receptor, the vitronectinreceptor and the platelet glycoprotein IIb) ((Corbi, A. et al., EMBO J.6:4023-4028 (1987)),12-14), consensus sequences were derived for theregions flanking the putative divalent cation-binding sites. Consensusresidues were defined as those appearing in at least 30% of the analyzedsequences. The consensus divalent cation-binding sequence and thecoordination axes of the residues ligating the divalent cation are basedon the sequence and the 3-dimensional structure of the Ca²⁺ and Mg²⁺-binding proteins parvalbumin, troponin C and calmodulin (38) and areshown below the alignments of the seven repeats.

FIGS. 4A-4J show a comparison of the primary structure of the α-subunitsof Mac-1, p150,95, vitronectin receptor, fibronectin receptor, andglycoprotein IIb. Identities between Mac-1 and the rest of the integrinα-subunits are boxed. The putative divalent cation-binding sites and theconversed flanking sequences are underlined by continuous and dottedlines, respectively. The putative transmembrane regions are indicated byTM.

FIGS. 5A-5B show a comparison of the sequences of the leukocyte specificdomain of Mac-1 alpha-subunit and p150,95 alpha-subunit with the Arepeats of von Willebrand factor, complement component C2 and factor B.Common residues between the sequences of Mac-1 α and/or p150,95 α andthe rest of the proteins are boxed. The alignments were performed asdescribed under Experimental Procedures.

FIG. 6 shows the proposed evolutionary relationsips of the Mac-1α-subunit, the A domains of von Willebrand factor, factor B and C2. Thebinding domains and the ligands for von Willebrand factor are indicatedon top (Shelton-Inloes, B. B. et al., Biochem. 25:3164-3171 (1986);Girma, J. P. et al., Blood 70:605-611 (1987)). The structures of factorB and C2 are as previously described (Mole, J. E. et al., J. Biol. Chem.259:3407-3412 (1984); Bentley, D. R., Biochem. J. 239:339-345 (1986))were also examined. RCA (Regulator of complement activation) repeats infactor B and C2 are 60 amino acids homologous repeats common to manycomplement components and receptors. The N-glycosylation sites and thecysteine residues in the α-subunit of Mac-1 are shown on top and bottomof its schematic representation, respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS I. The Nature of the LeukocyteAdhesion Proteins of the Mac-1 Family

The three leukocyte adhesion proteins Mac-1, p150,95, and LFA-1 differin function and in expression on leukocyte subpopulations. Mac-1 andp150,95 are expressed on neutrophils, and monocytes (Springer, T. A., etal., In: Biochemistry of Macrophages (CIBA Symposium 118); Pitman,London, pp. 102-126 (1986)). During differentiation of blood monocytesinto tissue macrophages, expression of p150,95 is greatly increased andMac-1 expression is decreased (Schwarting, R., et al., Blood 65:974-983(1985); Hogg, N., et al., Eur. J. Immunol. 16:240-248 (1986)). p150,95is also expressed on certain types of activated T and B lymphocytes, butis not expressed on these cells in the blood (Kaligaris-Cappio, F., etal., Blood 66:1035-1042 (1985); Miller, L. J., et al., J. Immunol.137:2891-2900 (1986); Keizer, G. D., et al., J. Immunol. 138:3130-3136(1987)).

LFA-1 is present on all leukocytes except a subset of macrophages.Monoclonal antibody blocking studies have shown that LFA-1 is importantin T-lymphocyte-mediated killing, T helper lymphocyte responses, naturalkilling, and antibody-dependent killing (Springer, T. A., et al., Ann.Rev. Immunol. 5:223-252 (1987)). Adhesion to the target cell is a stepwhich is blocked by antibodies against LFA-1. Functional studies havesuggested that LFA-1 interacts with several ligands, one of which isICAM-1 (Rothlein, R., et al., J. Immunol. 137:1270-1274 (1986)).

Mac-1 and p150,95 are expressed in an intracellular, vesicularcompartment in circulating neutrophils and monocytes which is mobilizedto the cell surface by inflammatory mediators (Todd, R. F., et al., J.Clin. Invest. 74:1280-1290 (1984); Springer, T. A., et al., In:Biochemistry of Macrophages (CIBA Symposium 118), Pitman, London, pp.102-126 (1986); Lanier, L. L., et al., Eur. J. Immunol. 15:713-718(1985); Yancey, K. B., et al., J. Immunol. 135:465-470 (1985)). Thismobilization correlates with increased adhesiveness (Anderson, D. C., etal., Ann. Rev. Med. 38:175-194 (1987)). Mac-1 α-subunit message wasdetected in blood monocytes and PMA-induced myeloid cell lines, but notin cells of the T or B lineages, correlating with Mac-1 protein surfaceexpression.

Some cytotoxic T lymphocyte clones have been found to express similarquantities of p150,95 and LFA-1. Monoclonal antibodies to the LFA-1 andp150,95 alpha-subunits inhibit killing by such CTL clones to similarextents and are additive in their inhibitory effects (Keizer, G. D., etal., J. Immunol. 138:3130-3136 (1987)). Furthermore, antibodies top150,95 alpha-subunits have been shown to inhibit monocyte attachment toendothelium (Keizer, G. D., et al., Eur. J. Immunol. 11:1317-1322(1987)).

Monoclonal antibodies to Mac-1 or p150,95 Inhibit neutrophil aggregationand adherence to endothelial cells, protein-coated surfaces, bacteria,protozoan parasites, and fungi (Harlan, J. M., et al., Blood 6:167-178(1985); Springer, T. A., at al., In: Biochemistry of Macrophages (CIBASymposium 118), Pitman, London, pp. 102-126 (1986); Dana, N., et al., J.Immunol. 137:3259 (1986); Bullock, W. D., et al., J. Exper. Med.165:195-210 (1987); Mosser, D. M., et al., J. Immunol, 135:2785-2789(1985)).

Mac-1 (CD 11b/CD18) is a leukocyte adhesion heterodimeric glycoproteinwhich functions as a receptor for iC3b (CR3) in addition to its role incell-cell and cell-substrate adhesive interactions (Beller, D. I., etal., J. Exper. Med. 1561000-1009 (1982)). Detergent-soluble Mac-1 andp150,95 have been shown to be able to bind to iC3b-Sepharose (Hicklem,K. J., et al., Biochem. J. 231:233-236 (1985)).

The α-subunit of Mac-1 is a transmembrane protein of 1137 residues witha long extracellular domain (1092 residues) and a 19-amino acidcytoplasmic tail. The extracellular domain contains 3 putative divalentcation-binding sequences and 19 potential N-glycosylation sites. Theamino acid sequence of Mac-1 α shows that it is a member of the integrinsuperfamily; Mac-1 α shows 63% identity to the α-subunit of theleukocyte adhesion glycoprotein p150,95 and 25% to the α-subunits of theextracellular matrix receptors platelet glycoprotein IIb/IlIa, thefibronectin receptor and the vitronectin receptor. The Mac-1 α-subunitputative divalent cation-binding sites and the flanking regions exhibita high degree of Identity both to the p150,95 α-subunit (87% identity atthe amino acid level) and to the rest of the integrin α-subunits (38%).The α-subunit of Mac-1, like the p150,95 α-subunit, contains a domain of187 amino acids in the extracellular region which is absent in otherintegrins. This leukocyte or "L" domain is homologous to the A domainsof van Willebrand factor, which in turn are homologous to regions of theC3-binding proteins factor B and C2. These findings draw attention tothis region of Mac-1 as a potential binding site for iC3b.

The functional role of Mac-1 was first illustrated by the ability ofanti-Mac-1 α-subunit monoclonal antibodies (MAb) to block the rosettingof iC3b-coated erythrocytes to macrophages and polymorphonuclearleukocytes (Beller, D. I. et al., J. Exper. Med. 156:1000-1009 (1982)),demonstrating that Mac-1 is indistinguishable from the complementreceptor type three (CR3). Subsequently, the involvement of Mac-1 ininflammatory processes was evidenced by the inhibition of neutrophilaggregation and adhesion to endothelial cells by anti-Mac-1 α-subunitand anti-β-subunit-specific MAb (Anderson, D. C. et al., J. Immunol.137:15-27 (1986); Dana, N. et al., J. Immunol. 137:3259-3263 (1986);Vedder, N. B. et al., J. Clin. Invest. 81:672-682 (1988)). Recentepitope mapping studies have suggested that the sites involved iniC3b-binding are distinct from those involved in neutrophil aggregationand adherence to protein-coated plastic (Anderson, D. C. et al., J.Immunol. 137:15-27 (1986); Dana, N. et al., J. Immunol. 137:3259-3263(1986), Rosen, H. et al;, J. Exper. Med. 166:1685-1701 (1987)).Therefore, Mac-1 appears to be a multivalent receptor with at least twoindependent adhesion-related functions.

The expression of functional activity of Mac-1 is regulated duringleukocyte differentiation and activation. Differentiation and maturationof myelomonocytic cell lines results in increased Mac-1 expression(Miller, L. J. et al., J. Immunol. 137:2891-2900 (1986)), while bloodmonoctye differentiation into tissue macrophages is accompanied by aconsiderable decrease in the amount of Mac-1 on all cell surface (Hogg,N. et al., Eur. J. Immunol. 16:240-248 (19886)). The expression of Mac-1on the surface of circulating meutrophils and monocytes is upregulatedby inflammatory stimuli; Mac-1 is stored in an intracellular vesicularcompartment which is rapidly mobilized to the cell surface bychemoattractants (Todd, R. F. et al., J. Clin. Invest. 74:1280-1290(1984)); Miller, L. J. et al., J. Clin. Invest. 80:535-544 (1987)).Although the augmented expression of Mac-1 can lead to increasedadhesiveness, qualitative changes after cell activation may also beimportant in regulation ligand binding (Detmers, P. A. et al., J. CellBiol. 105:1137-1145 (1987)). Both the qualitative and quantitativechanges may be important in regulation of leukocyte binding topost-capillary endothelium at inflammatory sites.

The N-terminal sequence of the murine and human Mac-1 α-subunits(Miller, L. J. et al., J. Immunol. 138:2381-2383 (1987); Springer, T. A.et al., Nature 314:540-542 (1985)) and a murine genomic clone encoding ashort N-terminal exon (Sastre, L. et al., Proc. Natl. Acad. Sci.(U.S.A.) 83:5644-5648 (1986)) have been reported.

Most of the Mac-1 α-subunit is similar to the α-subunits of theextracellular matrix receptor integrins, with an additional domain whichis related to the A repeats of von Willebrand factor and to twoC3-binding proteins, Factor B and C2.

II. Cloning of the Mac-1 Alpha-subunit

Any of a variety of methods may be used to clone the Mac-1 alpha-subunitgene. One such method entails analyzing a shuttle vector library of cDNAinserts (derived from a Mac-1 alpha-subunit expressing cell) for thepresence of an insert which contains the Mac-1 alpha-subunit gene. Suchan analysis may be conducted by transfecting cells with the vector, andthen assaying for Mac-1 alpha-subunit expression. Mac-1 alpha-subunit ispreferably assayed using antibodies specific for the Mac-1alpha-subunit. A preferred method for cloning the Mac-1 alpha-subunitgene entails determining the amino acid sequence of the Mac-1alpha-subunit molecule, or tryptic peptides of the molecule. Toaccomplish this task, Mac-1 alpha-subunit molecules are preferablypurified from producer cells by monoclonal antibody affinitychromatography and isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis ("SDS-PAGE") andelectroelution (Miller, L. J., et al., J. Immunol. 138:2381-2383 (1987),which reference herein is incorporated by reference). The alpha-subunitmolecules are fragmented as with cyanogen bromide, or with proteasessuch as papain, chymotrypsin, or trypsin (Oike, Y., et al., J. Biol.Chem. 257:9751-9758 (1982); Liu, C., et al., Int. J. Pept. Protein Res.21:209-215 (1983)). Preferably, the alpha-subunit is proteolyticallydigested with trypsin. The resulting peptides are separated byreverse-phase HPLC and subjected to amino acid sequencing. To accomplishthis task, the protein is, preferably, analyzed by automatedsequenators. Although it is possible to determine the entire amino acidsequence of the Mac-1 alpha-subunit, it is preferable to determine thesequence of peptide fragments of the molecule. A preferred source of theMac-1 alpha-subunit is the SKW3 cell line.

The sequence of amino acid residues in a peptide is designated hereineither through the use of their commonly employed three-letterdesignations or by their single-letter designations. A listing of thesethree-letter and one-letter designations may be found in textbooks suchas Biochemistry, Lehninger, A., Orth Publishers, New York, N.Y. (1970).When such a sequence is listed vertically, the amino terminal residue isintended to be at the top of the list, and the carboxy terminal residueof the peptide is intended to be at the bottom of the list. Similarly,when listed horizontally, the amino terminus is intended to be on theleft end whereas the carboxy terminus is intended to be at the rightend.

The residues of amino acids in a peptide may be separated by hyphens.Such hyphens are intended solely to facilitate the presentation of asequence. As a purely illustrative example, the amino acid sequencedesignated:

    -Gly-Ala-Ser-Phe-

indicates that an Ala residue is linked to the carboxy group of Gly, andthat a Ser residue is linked to the carboxy group of the Ala residue andto the amino group of a Phe residue. The designation further indicatesthat the amino acid sequence contains the tetrapeptide Gly-Ala-Ser-Phe.The designation is not intended to limit the amino acid sequence to thisone tetrapeptide, but is intended to include (1) the tetrapeptide havingone or more amino acids linked to either its amino or carboxy end, (2)the tetrapeptide having one or more amino acid residues linked to bothits amino and its carboxy ends, (3) the tetrapeptide having noadditional amino acid residues.

Once one or more suitable peptide fragments have been sequenced, the DNAsequences capable of encoding them are examined. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid (Watson, J. D., In: Molecular Biology of the Gene,3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977), pp. 356-357).The peptide fragments are analyzed to identify sequences of amino acidswhich may be encoded by oligonucleotides having the lowest degree ofdegeneracy. This is preferably accomplished by identifying sequencesthat contain amino acids which are encoded by only a single codon.

Although occasionally an amino acid sequences may be encoded by only asingle oligonucleotide, frequently the amino acid sequence may beencoded by any of a set of similar oligonucleotides. Important, whereasall of the members of this set contain oligonucleotides which arecapable of encoding the peptide fragment and, thus, potentially containthe same oligonucleotide sequence as the gene which encodes the peptidefragment, only one member of the set contains the nucleotide sequencethat is identical to the nucleotide sequence of the gene. Because thismember is present within the set, and is capable of hybridizing to DNAeven in the presence of the other members of the set, it is possible toemploy the unfractionated set of oligonucleotides in the same manner inwhich one would employ a single oligonucleotide to clone the gene thatencodes the peptide.

A suitable oligonucleotide, or set of oligonucleotides, which is capableof encoding a fragment of the Mac-1 alpha-subunit gene (or which iscomplementary to such an oligonucleotide, or set of oligonucleotides) isidentified (using the above-described procedure), synthesized, andhybridized by means well known in the art, against a DNA or, morepreferably, a cDNA preparation derived from human cells which arecapable of expressing the Mac-1 alpha-subunit gene. Techniques ofnucleic acid hybridization are disclosed by Maniatis, T., et al. (In:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories,Cold Spring Harbor, N.Y. (1982)), and by Haymes, B. D., et al. (In:Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,D.C. (1985)), which references are herein incorporated by reference. Thesource of DNA or cDNA used will preferably have been enriched for Mac-1alpha-subunit sequences. Such enrichment can most easily be obtainedfrom cDNA obtained by extracting RNA from cells which produce highlevels of the Mac-1 alpha-subunit.

Techniques such as, or similar to, those described above havesuccessfully enabled the cloning of genes for human aldehydedehydrogenases (Hsu, L. C., et al., Proc. Natl. Acad. Sci. USA82:3771-3775 (1985)), fibronectin (Suzuki, S., et al., Eur. Mol. Biol.Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter,P., et al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-typeplasminogen activator (Pennica, D., et al., Nature 301:214-221 (1983))and human term placental alkaline phosphatase complementary DNA (Kam,W., et al., Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985)).

Using the genetic code (Watson, J. D., In: Molecular Biology of theGene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977)), one ormore different oligonucleotides can be identified, each of which wouldbe capable of encoding the Mac-1 alpha-subunit tryptic peptides. Theprobability that a particular oligonucleotide will, in fact, constitutethe actual Mac-1 alpha-subunit-encoding sequence can be estimated byconsidering abnormal base pairing relationships and the frequency withwhich a particular codon is actually used (to encode a particular aminoacid) in eukaryotic cells. Such "codon usage rules" are disclosed byLathe, R., et al., J. Molec. Biol. 183:1-12 (1985). Using the "codonusage rules" of Lathe, a single oligonucleotide, or a set ofoligonucleotides, that contains a theoretical "most probable" nucleotidesequence capable of encoding the Mac-1 alpha-subunit tryptic peptidesequences is identified.

The oligonucleotide, or set of oligonucleotides, containing thetheoretical "most probable" sequence capable of encoding the Mac-1alpha-subunit fragments is used to identify the sequence of acomplementary oligonucleotide or set of oligonucleotides which iscapable of hybridizing to the "most probable" sequence, or set ofsequences. An oligonucleotide containing such a complementary sequencecan be employed as a probe to identify and isolate the Mac-1alpha-subunit gene (Maniatis, T., et al., Molecular Cloning A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982).

Thus, in summary, the actual identification of Mac-1 alpha-subunitpeptide sequences permits the identification of a theoretical "mostprobable" DNA sequence, or a set of such sequences, capable of encodingsuch a peptide. By constructing an oligonucleotide complementary to thistheoretical sequence (or by constructing a set of oligonucleotidescomplementary to the set of "most probable" oligonucleotides), oneobtains a DNA molecule (or set of DNA molecules), capable of functioningas a probe to identify and isolate the Mac-1 alpha-subunit gene.

Single stranded oligonucleotide molecules complementary to the "mostprobable" Mac-1 alpha-subunit tryptic peptide encoding sequences weresynthesized using procedures which are well known to those of ordinaryskill in the art (Belagaje, R., et al., J. Biol. Chem. 254:5765-5780(1979); Maniatis, T., et al., In: Molecular Mechanisms in the Control ofGene Expression, Nierlich, D. P., et al., Eds., Acad. Press, NY (1976);Wu, R., et al., Prog. Nucl. Acid Res. Molec. Biol. 21:101-141 (1978);Khorana, R. G., Science 203:614-625 (1979)). Additionally, DNA synthesismay be achieved through the use of automated synthesizers.

It is possible to clone the Mac-1 alpha-subunit gene from eukaryotic DNApreparations suspected of containing this gene. To identify and clonethe gene which encodes the Mac-1 alpha-subunit protein, a DNA, or morepreferably a cDNA, library is screened for its ability to hybridize withthe oligonucleotide probes described above. Suitable DNA preparations(such as human genomic DNA) are enzymatically cleaved, or randomlysheared, and ligated into recombinant vectors. The ability of theserecombinant vectors to hybridize to the above-described oligonucleotideprobes is then measured. Procedures for hybridization are disclosed, forexample, in Maniatis, T., Molecular Cloning A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1982) or in Haymes, B.T., et al., Nucleic Acid Hybridization a Practical Approach, IRL Press,Oxford, England (1985). Vectors found capable of such hybridization arethen analyzed to determine the extent and nature of the Mac-1alpha-subunit sequences which they contain. Based purely on statisticalconsiderations, a gene such as that which encodes the Mac-1alpha-subunit molecule could be unambiguously identified (viahybridization screening) using an oligonucleotide probe having only 18nucleotides.

The cloned Mac-1 alpha-subunit gene, obtained through the methoddescribed above, may be operably linked to an expression vector, andintroduced into prokaryotic or eukaryotic cells to produce the Mac-1alpha-subunit protein. Techniques for such manipulations are disclosedby Maniatis, T., et al., supra, and are well known in the art.

III. The Expression of the Mac-1 Alpha-subunit

The present invention derives, in part, from the discovery of the cDNAsequence which encodes the alpha-subunit of the Mac-1 molecule. Byoperably linking this sequence (or a fragment of this sequence) to afunctional promoter, it is possible to direct the expression of thealpha-subunit of Mac-1 (or a functional derivative thereof) in a cell,or organism.

A nucleic acid molecule, such as DNA, is said to be "capable ofexpressing" a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are "operably linked" to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression. The precise natureof the regulatory regions needed for gene expression may vary fromorganism to organism, but shall in general include a promoter regionwhich, in prokaryotes, contains both the promoter (which directs theinitiation of RNA transcription) as well as the DNA sequences which,when transcribed into RNA, will signal the initiation of proteinsynthesis. Regulatory regions in eukaryotic cells will in generalinclude a promoter region sufficient to direct the initiation of RNAsynthesis.

Two DNA sequences (such as a promoter region sequence and a Mac-1alpha-subunit-encoding sequence) are said to be operably linked if thenature of the linkage between the two DNA sequences does not (1) resultin the introduction of a frame-shift mutation, (2) interfere with theability of the promoter region sequence to direct the transcription ofthe Mac-1 alpha-subunit-encoding sequence, or (3) interfere with theability of the Mac-1 alpha-subunit-encoding sequence to be transcribedby the promoter region sequence. Thus, a promoter region would beoperably linked to a DNA sequence if the promoter were capable ofeffecting transcription of that DNA sequence.

The present invention encompasses the expression of the Mac-1alpha-subunit (or a functional derivative thereof) in either prokaryoticor eukaryotic cells. To express the Mac-1 alpha-subunit (or a functionalderivative thereof) in a prokaryotic cell (such as, for example, E.coli, B. subtilis, Pseudomonas, Streptomyces, etc.), it is necessary tooperably link the Mac-1 alpha-subunit-encoding sequence to a functionalprokaryotic promoter. Such promoters may be either constitutive or, morepreferably, regulatable (i.e., inducible or derepressible). Examples ofconstitutive promoters include the int promoter of bacteriophage λ, thebla promoter of the β-lactamase gene of pBR322, and the CAT promoter ofthe chloramphenicol acetyl transferase gene of pPR325, etc. Examples ofinducible prokaryotic promoters include the major right and leftpromoters of bacteriophage λ (P_(L) and P_(R)), the trp, recA, lacZ,lacI, and gal promoters of E. coli, the α-amylase (Ulmanen, I., et al.,J. Bacteriol. 162:176-182 (1985)) and the σ-28-specific promoters of B.subtilis (Gilman, M. Z., et al., Gene 32:11-20 (1984)), the promoters ofthe bacteriophages of Bacillus (Gryczan, T. J., In: The MolecularBiology of the Bacilli, Academic Press, Inc., NY (1982)), andStreptomyces promoters (Ward, J. M., et al., Mol. Gen. Genet.203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B. R.,(J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442(1984)).

Proper expression in a prokaryotic cell requires the presence of aribosome binding site upstream of the gene-encoding sequence. Suchribosome binding sites are disclosed, for example, by Gold, L., et al.(Ann. Rev. Microbiol. 35:365-404 (1981)).

If expression is desired in a eukarotic cell, such as yeast, fungi,mammalian cells, or plant cells, then it shall be necessary to employ apromoter capable of directing transcription in such a eukaryotic host.Preferred eukaryotic promoters include the promoter of the mousemetallothionein I gene (Hamer, D., et al., J. Mol. Appl. Gen. 1:273-288(1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365(1982)); the SV40 early promoter (Benoist, C., et al., Nature (London)290:304-310 (1981)); the yeast gal4 gene promoter (Johnston, S. A., etal., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P. A., etal., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the Mac-1 alpha-subunit (or a functionalderivative thereof) does not contain any intervening codons which arecapable of encoding a methionine (i.e., AUG). The presence of suchcodons results either in a formation of a fusion protein (if the AUGcodon is in the same reading frame as the Mac-1 encoding DNA sequence)or a frame-shift mutation (if the AUG codon is not in the same readingframe as the Mac-1 encoding sequence).

A DNA sequence which encodes the Mac-1 protein (or a functionalderivative thereof) when operably linked to a functional promoter ispreferably introduced into a recipient cell by any of a variety ofsuitable means: transformation, transfection, conjugation, protoplastfusion, electroporation, etc.

The Mac-1 alpha-subunit-encoding sequence and an operably linkedpromoter may be introduced into a recipient cell either as anon-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the Mac-1 alpha-subunit polypeptide may occur through the transientexpression of the introduced sequence. Alternatively, permanentexpression may occur through the integration of the introduced sequenceinto the host chromosome.

Preferably, the introduced sequence will be incorporated into a plasmidor viral vector capable of autonomous replication in the recipient host.Any of a wide variety of vectors may be employed for this purpose.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to"shuttle" the vector between host cells of different species. Preferredprokaryotic vectors include plasmids such as those capable ofreplication in E. coli (such as, for example, pBR322, ColE1, pSC101,pACYC 184, πVX. Such plasmids are, for example, disclosed by Maniatis,T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1982)). Bacillus plasmidsinclude pC194, pC221, pT127, etc. Such plasmids are disclosed byGryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press,NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJ101(Kendall, K. J., et al., J. Bacteriol. 169:4177-4183 (1987)), andstreptomyces bacteriophages such as φC31 (Chater, K. F., et al., In:Sixth International Symposium on Actinomyceteles Biology, AkademiaiKaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids arereviewed by John, J. F., et al. (Rev. Infect. Dis. 8:693-704 (1986)),and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 (1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-microncircle, etc., or their derivatives. Such plasmids are well known in theart (Botstein, D., et al., Miami Wntr. Symp. 19:265-274 (1982); Broach,J. R., In: The Molecular Biology of the Yeast Saccharomyces: Life Cycleand Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., p. 445-470 (1981); Broach, J. R., Cell 28:203-204 (1982); Bollon,D. P., et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, T.,In: Cell Biology: A Comprehensive Treatise, Vol. 3. Gene Expression,Academic Press, NY, pp. 563-608 (1980)).

IV. Uses of the Mac-1 Alpha-subunit, or Fragments Thereof

The present invention provides the nucleic acid and protein sequences ofthe alpha-subunit of the Mac-1 receptor molecule. This discovery permitsthe use of recombinant DNA technology to produce the Mac-1 alpha-subunitmolecule. As discussed further below, one embodiment of the presentinvention pertains to the use of the alpha-subunit of the Mac-1 moleculeby itself, as an anti-inflammatory agent. In a preferred embodiment, thealpha-subunit of the Mac-1 molecule will be used in combination with itsbeta-subunit. Most preferably in a combination which creates afunctional Mac-a alpha and beta heterodimer. Most preferably, suchheterodimers will container recombinant Mac-1 alpha subunits or theirfunctional derivatives. Such a combination may be produced using avariety of methods. For example, the beta-subunit of Mac-1 may beproduced separately from the Mac-1 alpha-subunit, and the two moleculesmay then be mixed together. It is, however, preferable to produce boththe alpha and beta-subunits of Mac-1 in the same host cell in order tofacilitate their self-assembly into the Mac-1 heterodimer receptormolecule. The beta-subunit of Mac-1 (which is common to LFA-1, andp150,95) may be produced either by chemical synthesis, or by recombinantDNA techniques (Kishimoto, T. K., et al., Cell 48:681-690 (1987)). Thecloning of the beta-subunit of Mac-1 is further disclosed in commonlyassigned, co-pending U.S. patent application Ser. No. 019,440, filed onFeb. 26, 1987, which application is herein incorporated by reference.

One aspect of the present invention relates to the discovery of thenucleic acid and protein sequences of the alpha-subunit of the Mac-1receptor molecule. This discovery permits the use of recombinant DNAtechnology to produce functional derivatives of the Mac-1 alpha-subunitwhich may function as antagonists of cellular adhesion. As used herein,an "antagonist of cellular adhesion" is meant to refer to any moleculecapable of inhibiting the process of cell-cell or cell-substrateadhesion. It is possible to determine whether a particular compound isan antagonist by performing an assay of monocyte adhesion to endothelialcells, neutrophil aggregation, or iCb3 rosetting of neutrophils.Suitable assays of cellular adhesion are disclosed, for example, byAnderson, D. C., et al. (J. Immunol. 137:15-27 (1986)) and by Keizer, G.D., et al. (Eur. J. Immunol. 17:1317-1322 (1987)) which references areherein incorporated by reference. Antagonists of cellular adhesion maybe employed as anti-inflammatory agents.

As used herein, a "functional derivative" of the alpha-subunit of Mac-1is a compound which possesses a biological activity (either functionalor structural) that is substantially similar to a biological activity ofthe alpha-subunit of Mac-1. Examples of biological activities includethe ability to bind to the natural ligand of Mac-1, or well on theability to bind to the β-subunit of the LFA family of glycoproteins.Such binding would inhibit adhesion related events such as granulocytemigration through endothelium, granulocyte aggregation and iCb3rossetting. A molecule is said to be "substantially similar" to anothermolecule if both molecules have substantially similar structures or ifboth molecules possess a similar biological activity. The "functionalderivatives" of the alpha-subunit of Mac-1 include both "fragments" and"variants" of the Mac-1 alpha-subunit. The term "fragment of thealpha-subunit of Mac-1" is meant to refer to any polypeptide subset ofthat molecule. The term "variant of the alpha-subunit of Mac-1" is meantto refer to a molecule substantially similar in structure to either theentire molecule, or to a fragment thereof provided that the "variant"has at least one biological activity that is either similar to anactivity of the alpha-subunit of Mac-1 or inhibitory to an activity ofMac-1. Thus, provided that a molecule possesses at least one biologicalactivity that is either similar to an activity of Mac-1 or inhibitory tosuch an activity, it is considered a "variant" of the alpha-subunitMac-1, as that term is used herein, even if one of the moleculescontains one or more amino acids not found in the other, or if thesequences of amino acid residues in the two molecules are not identical.Thus, for example, a compound lacking (or containing) one or more aminoacid residues found (or not found) in the alpha-subunit of Mac-1 wouldbe considered to be a variant of the alpha-subunit of Mac-1 if thatcompound possessed a biological activity similar to (or inhibitory to) abiological activity of the alpha-subunit of Mac-1. The term "biologicalactivity" is intended to encompass "catalytic" as well as "structural"activity (i.e., the capacity to bind to another molecule, such as thebeta-subunit of Mac-1, anti-alpha-subunit Mac-1 antibody, iCb3, oranother natural ligand of Mac-1), etc.

The present invention provides a method for producing functionalderivatives of the alpha-subunit of the Mac-1 molecule. To obtain suchderivatives, it is necessary only to mutagenize a DNA, RNA, or (morepreferably) the cDNA sequence which encodes the Mac-1 alpha-subunit.Mutagenesis can either be random, or site specific. Further, mutagenesismay either be spontaneous or induced using chemical, radioactive, orrecombinant techniques.

The scope of the present invention is further intended to includefunctional derivatives which lack certain amino acid residues, or whichcontain altered amino acid residues, so long as such derivatives exhibitthe capacity to enhance or inhibit cellular adhesion.

Chemical mutagens include base analogs (such as, for example,5-bromouracil, or 2-aminopurine); deaminating agents (such as, forexample, nitrous acid, hydroxylamine, etc.); alkylating agents (such as,for example, methyl methanesulphonate, nitrosoguanidine, etc.); orintercolating agents (such as, for example, acridine orange, ethidiumbromide, psoralen, etc.). Radiation-induced mutation can be caused byagents such as ultraviolet light, gamma, X-ray, etc. Techniques formutagenizing nucleic acid molecules may be found in Miller, J. H. (In:Experiments in Molecular Biology, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1972)), and Silhavy, T. J., et al. (In: Experimentswith Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1984)).

Site-specific mutagenesis may be employed to produce specific mutationsat desired sites of the nucleic acid encoding the Mac-1 alpha-subunit.In brief, such procedures generally entail the synthesis of a syntheticoligonucleotide having a desired and defined DNA sequence. Methods forsynthesizing such oligonucleotides are disclosed by Itakura, K., et al.(Ann. Rev. Biochem. 53:323-356 (1984)). A nucleic acid molecule whichencodes the Mac-1 alpha-subunit protein, or a functional derivativethereof, is generally subcloned onto a double-stranded vector such asM13, φX174, etc., whose single strands may be separated one fromanother. A single strand of the vector is then incubated in the presenceof the synthetic oligonucleotide. Since the DNA of the oligonucleotideis controllably defined, it is possible to construct an oligonucleotidecapable of base pairing with any region of the Mac-1alpha-subunit-encoding nucleic acid. Once base pairing has occurredbetween the oligonucleotide and the single-stranded plasmid, it ispossible to extend the oligonucleotide using DNA polymerase to create adouble-stranded DNA molecule which may then be sealed by DNA ligase.When this double-stranded DNA molecule is introduced into a cell,semi-conservative DNA replication will result in the production ofprogeny molecules in which the DNA sequence of the oligonucleotidefragment has been incorporated into the Mac-1 alpha-subunit-encodingsequences.

The Mac-1 alpha subunit of the present invention, or its functionalderivatives, may alternatively be prepared by synthetic chemical methodusing the well-known Merriefield or other techniques of peptidesynthesis. Alternatively, such molecules may also be prepared bychemical synthesis of nucleic acid molecules (using, for example,phosphodiester synthesis techniques), which, upon expression, willresult in their production.

Thus, if one desired to introduce a point mutation, and exogenous DNAsequence into a specific site in the Mac-1-encoding sequence, or tocreate a deletion of nucleotides normally present in such a sequence,one would design an oligonucleotide fragment which contained (or lacked)the mutation or sequence, and then pursue the above-described procedure.In order to introduce such a mutation or exogenous DNA sequence into aparticular region of the Mac-1 alpha-subunit-encoding nucleic acid, itis necessary to surround the mutation or the exogenous DNA sequence withflanking DNA sequences that are complementary to the DNA sequence of theregion whose mutagenesis is desired. (Jenkins, F., et al., Bioessays5:244-247 (1986); Doerfler, W., Angew. Chem. Int. Ed. Engl. 23:919-931(1984); Kaina, B., Biol. Zentralbl. 99:513-531 (1980); Kunkel, Proc.Natl. Acad. Sci. (USA) 82:488-492 (1985); Nisbet, I. T., et al., GeneAnal. Tech. 2:23-29 (1985); Hines, J. C., et al., Gene 11:207-218(1980); Messing, J., et al., Nucl. Acid. Res. 9:309 (1981)).

Mutations can also be produced through the application of recombinantDNA technology. For example, the nucleotide sequence of a nucleic acidmolecule which encodes the Mac-1 alpha-subunit can be scanned toidentify oligonucleotide sites which are recognizable by restrictionendonucleases. Such endonucleases can then be used to specificallycleave the nucleic acid sequence at a recognized site. By using arestriction endonuclease that recognizes (and cleaves at) two positionsin the Mac-1-encoding sequence, it is possible to excise a fragment ofthe Mac-1 alpha-subunit-encoding sequence. Alternatively, it is possibleto use two different endonucleases for this purpose. By incubating thecleaved molecules in the presence of DNA ligase, it is possible toreseal the Mac-1 alpha-subunit-encoding sequences to form a singlesequence (which lacks the excised fragment). If no appropriaterestriction endonuclease recognition sites exist in the Mac-1alpha-subunit-encoding sequences, then such sites may be introduced intothe sequences by the site-specific mutagenesis procedure describedabove.

Mutations may alternatively be introduced by cleaving the Mac-1alpha-subunit-encoding sequence and "nibbling" the free termini with anexonuclease. By such treatment it is possible to introduce not onlydeletions, but frame-shift and other types of mutations. This techniqueis, moreover, capable of introducing novel restriction endonucleasesites into the Mac-1 alpha-subunit-encoding sequence. Methods for usingrestriction endonucleases, DNA ligases, and exonucleases are disclosed,for example, by Maniatis, T., et al. (In: Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982)).

Recombinant DNA techniques may also be used to produce fusion proteinscomposed of the Mac-1 alpha-subunit protein (or a functional derivativethereof) and a novel polypeptide. This novel polypeptide is not limitedto any particular polypeptide and may comprise either a single aminoacid or any set or permutation of amino acids. Such fusion molecules maybe produced by ligating a DNA sequence which encodes the novelpolypeptide to a DNA sequence which encodes the Mac-1 alpha-subunit (ora functional derivative thereof), in a manner which does not introduce aframe-shift mutation. Examples of preferred polypeptides which may befused to the Mac-1 alpha-subunit gene (or a functional derivativethereof) include eukaryotic or prokaryotic signal sequences (Gilbert,W., et al., U.S. Pat. No. 4,411,994; Casadaban, M., et al., Proc. Natl.Acad. Sci. (USA) 76:4530-4533 (1979)), or polypeptides which extend (ordiminish) the stability, biological half-life, or potency of the Mac-1alpha-subunit (or a functional derivative thereof). An excellent reviewof the methodology of gene fusions is provided by Silhavy, T. J., et al.(In: Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984)).

Antibodies (especially monoclonal antibodies) may be elicited inresponse to immunization with fragments of the Mac-1 alpha-subunit or tothe recombinant Mac-1 alpha subunit. Such antibodies can be used toprevent the binding of some leukocytes to endothelial cells, and thusmay be employed as anti-inflammatory agents.

Using the methods described above, fragments of the Mac-1 alpha-subunitmay be prepared and assayed to determine whether they are antagonists ofcellular adhesion. Fragments found to be antagonists of cellularadhesion may be employed as anti-inflammatory agents in accordance withthe present invention.

The present invention derives in part from the discovery that theadhesion ability of circulating neutrophils and monocytes results frominteractions involving the Mac-1 receptor molecule. Since cellularadhesion is required in order that such cells may migrate to sites ofinflammation and/or carry out various effector functions contributing toinflammation, agents which inhibit such cellular adhesion will attenuateor prevent inflammation. The Mac-1 receptor molecule is present on thesurface of neutrophils and monocyte cells. The adhesion of these cellsto plastic surfaces, or to monolayers of endothelial cells, is mediatedby the Mac-1 receptor molecule. In addition, the ability of monocytes tophagocytose foreign material has been found to be mediated by the Mac-1receptor molecule. The receptor molecule has also been implicated ashaving a role in chemokinesis, and chemotaxis of monocytes.

Agents which interfere with the capacity of the Mac-1 receptor moleculeto bind to its natural binding ligand are thus capable of impairing allof the above-described Mac-1-dependent functions. Hence, these agentsmay serve as anti-inflammatory agents in accordance with the presentinvention. Such agents include the Mac-1 alpha-subunit, Mac-1 (i.e., theheterodimer composed of alpha and beta-subunits), a composition composedof the non-associated Mac-1 alpha and beta subunits, and antibodycapable of binding to the Mac-1 alpha-subunit, or to fragments of thatsubunit. In one embodiment of the present invention, such agents arecomposed of soluble Mac-1 alpha subunits or soluble functionalderivatives thereof, and are able to interfere with or inhibit eitherthe binding of natural Mac-1 alpha with its beta subunit, or inhibit theability of Mac-1 to bind to any of its natural binding ligands. All ofsuch agents may be used in accordance with the present invention. Theanti-inflammatory agents of the present invention are capable oftreating inflammation caused by a reaction of the non-specific defensesystem.

A "non-specific defense system reaction" is a response mediated byleukocyte cells incapable of immunological memory. Such cells includelymphocytes and macrophages. As used herein, inflammation is said toresult from a response of the non-specific defense system, if theinflammation is caused by, mediated by, or associated with a reaction ofthe non-specific defense system. Examples of inflammation which result,at least in part, from a reaction of the non-specific defense systeminclude inflammation associated with conditions such as: asthma, adultrespiratory distress syndrome (ARDS) or multiple organ injury syndromessecondary to septicemia or trauma; reperfusion injury of myocardial orother tissues; acute glomerulonephritis; reactive arthritis; dermatoseswith acute inflammatory components; acute purulent meningitis or othercentral nervous system inflammatory disorders; thermal injury;hemodialysis; leukapheresis; ulcerative colitis; Crohn's disease;necrotizing enterocolitis; granulocyte transfusion associated syndromes;cytokine-induced toxicity; and atherosclerosis.

Since Mac-1 is expressed on cells which are capable of binding toendothelial tissue, the administration of the Mac-1 alpha-subunit, orMac-1 (alpha and beta-subunits) to a patient provides a means forimaging or visualizing endothelial tissue. Moreover, this procedureprovides diagnostic information concerning the quantity and distributionof the binding ligands of the Mac-1 receptor molecule which are presenton the visualized tissue. In such a use, the Mac-1 alpha-subunits (orMac-1 alpha beta receptor molecules) are detectably labeled, through theuse of radioisotopes, affinity labels (such as biotin, avidin, etc.)fluorescent labels, paramagnetic atoms, etc. Procedures foraccomplishing such labeling are well known to the art. The antibodies(or fragments thereof) may be detectably labeled through the use ofradioisotopes, enzyme labels, fluorescent labels, paramagnetic labels,electron-dense labels, toxin labels, etc. Preferred toxin labels includethe diphtheria toxin, ricin, and cholera toxins. The administration ofsuch labeled molecules into an individual will identify sites ofinflammation. Such detectable labels can also be used to assay thestatus of a patient's immune system. Clinical application of antibodiesin diagnostic imaging are reviewed by Grossman, H. B., Urol. Clin. NorthAmer. 13:465-474 (1986)), Unger, E. C. et al., Invest. Radiol.20:693-700 (1985)), and Khaw, B. A. et al., Science 209:295-297 (1980)).

The ability of monocytes to migrate spontaneously to sites ofinflammation is dependent upon Mac-1 (Keizer, G. D., et al., Eur. J.Immunol. 17:1317-1322 (1987)). Such migration may be inhibited byadministrating Mac-1 alpha-subunits, or Mac-1 (alpha and beta-subunit)to a patient.

Similarly, the ability of monocytoid cells to adhere to endothelialcells, and the ability of monocytoid cells to undergo chemotaxis,chemokinesis, or phagocytosis has been found to be dependent upon Mac-1(Keizer, G. D., et al., Eur. J. Immunol. 17:1317-1322 (1987)). Any ofthe anti-inflammatory agents of the present invention may be employed toinhibit such activates.

ICAMs (such as ICAM-1) are recognized by certain human viruses(particularly rhinoviruses of the major type (which bind to ICAM-1).These viruses bind to human cells by virtue of this recognition, andthereby mediate viral infection. Thus, a central step in the etiology ofviral disease is the interaction between these cellular receptors andthe virus.

Agents which suppress, compete with, or inhibit the ability of a virusto bind to an ICAM molecule thus have use in the treatment of viral (andparticularly rhinoviral) infection.

One aspect of the present invention thus concerns the ability of thealpha-subunit of MAC-1 and its functional derivatives to interact withICAM-1 and to thereby either prevent cell-viral attachment and viralinfection, or to attenuate or diminish the severity or duration of suchinfection.

Of particular interest to the present invention are functionalderivatives of the alpha-subunit of MAC-1 such as solubilized forms ofthe alpha-subunit of MAC-1, fragments of the alpha-subunit of MAC-1,etc. Such agents are preferably provided to a recipient patient as aheterodimer containing the molecule in association with a molecule ofthe beta-subunit of the CD-18 family. The above-described goal oftreating viral infection may be accomplished with a single agent or witha combination of more than one agents.

For the purpose of treating viral infection, the above-describedagent(s) of the present invention is to be provided to a recipientpatient (for example, by intranasal means) at a dosage sufficient topermit the agent(s) to suppress, compete with, or inhibit the ability ofa virus to bind to an ICAM molecule. Such a dosage shall, in general, be(for each agent provided) from 0.01 pg/kg patient weight to 1 mg/kgpatient weight, although greater or lesser amounts can be employed.

For the purpose of treating viral infection, the administration of suchagent(s) may be provided either "prophylactically" or "therapeutically."When provided prophylactically, the agent(s) are provided in advance of(i.e. prior to, at, or shortly after) the time of infection but inadvance of any symptoms of viral infection. The prophylacticadministration of the agent(s) serves to prevent or attenuate anysubsequent infection. When provided therapeutically, the agent(s) areprovided at (or shortly after) the onset of a symptom of actual viralinfection (such as, for example, the appearance of virally induced nasalcongestion, etc. or the detection of virus in bodily fluids, or thedetection of antibodies, directed against the virus, in the serum of aninfected patient, etc). The therapeutic administration of the agent(s)serves to attenuate any actual infection, and thus lessen its severityor duration.

V. Administration of the Mac-1 Alpha-Subunit

The therapeutic effects of Mac-1 alpha-subunit may be obtained byproviding to a patient the Mac-1 receptor molecule (α and β-subunits),the entire Mac-1 alpha-subunit molecule, or any therapeutically activefunctional derivative thereof. These molecules may be obtained eithersynthetically, or through the use of recombinant DNA technology.Fragments of the Mac-1 receptor or its alpha-subunit may additionally beobtained by proteolysis. The therapeutic advantages of these moleculesmay be augmented through the use of functional derivatives which possessadditional amino acid residues added to enhance coupling to carrier orto enhance activity.

The molecules of the present invention are said to be "substantiallyfree of natural contaminants" if preparations which contain them aresubstantially free of materials with which these products are normallyand naturally found.

In providing a patient with the therapeutic molecules of the presentinvention, the dosage of administered agent will vary depending uponsuch factors as the patient's age, weight, height, sex, general medicalcondition, previous medical history, etc. In general, it is desirable toprovide the recipient with a dosage of Mac-1 alpha-subunit (or afunctional derivative thereof) which is in the range of from about 1pg/kg to 10 mg/kg (body weight of patient), although a lower or higherdosage may be administered.

The molecules of the present invention may be administered to patientsintravenously, intramuscularly, subcutaneously, enterally, orparenterally. Administration may be by continuous infusion, or by singleor multiple boluses.

The anti-inflammatory agents of the present invention are intended to beprovided to recipient subjects in an amount sufficient to suppressinflammation. An amount is said to be sufficient to suppressinflammation if the dosage, route of administration, etc. of the agentare sufficient to attenuate or prevent inflammation. Theanti-inflammatory agents of the present invention may be provided eitherprior to the onset of inflammation (so as to suppress the anticipatedinflammation) or after the initiation of inflammation.

A composition is said to be "pharmacologically acceptable" if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a "therapeutically effective amount" if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient. The molecules of thepresent invention can be formulated according to known methods toprepare pharmaceutically acceptable compositions, whereby thesematerials, or their functional derivatives, are combined in admixturewith a pharmaceutically acceptable carrier vehicle. Suitable vehiclesand their formulation, inclusive of other human proteins, e.g., humanserum albumin, are described, for example, in Remington's PharmaceuticalSciences (16th ed.), Osol, A., ed., Mack, Easton, Pa. (1980). In orderto form a pharmaceutically effective composition suitable for effectiveadministration, such compositions will contain a therapeuticallyeffective amount of Mac-1 alpha-subunit, or its fragments or functionalderivatives, together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymers to complex or absorb Mac-1 alpha-subunit orits fragments or functional derivatives. The controlled delivery may beexercised by selecting appropriate macromolecules (for example,polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protaminesulfate) and the concentration of macromolecules as well as the methodsof incorporation in order to control release. Another possible method tocontrol the duration of action by controlled release preparations is toincorporate Mac-1 alpha-subunit molecules, their fragments, or theirfunctional derivatives, into particles of a polymeric material such aspolyesters, polyamino acids, hydrogels, poly(lactic acid), or ethylenevinylacetate copolymers. Alternatively, instead of incorporating theseagents into polymeric particles, it is possible to entrap thesemolecules in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly(methylmethacrylate)microcapsules, respectively, or in colloidal drug delivery systems, forexample, liposomes, albumin microspheres, microemulsions, nanoparticles,and nanocapsules in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences (1980).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 Protein Purification and Sequencing

The Mac-1 α/β complex was purified from leukocyte Triton X-100 lysatesby monoclonal antibody-affinity chromatography. Such purification wasaccomplished either through the use of the anti-Mac-1 α-subunitmonoclonal antibody, LM2/1 (Miller, L. J. et al., J. Immunol.138:2381-2383 (1987)), or, more preferably, through the use of anti-βmonoclonal antibody, IB4.5 (Wright, S. D. et al., Proc. Natl. Acad. Sci.(U.S.A.) 80:5699-5703 (1983)). Affinity chromatography was accomplishedby coupling the antibody to Sepharose as described by (Miller, L. J. etal., J. Immunol. 138:2381-2383 (1987); Schneider, C. et al., J. Biol.Chem. 257:10766-10769 (1982)).

Lysates of Mac-1 expressing cells were prepared according to the methodof (Miller, L. J. et al., J. Immunol. 138:2381-2383 (1987)). The lysatewas passed through a 3 ml protein A-Sepharose pre-column connected inseries with the 3.5 ml IB4.5 column after pre-washing of both columnswith 50 ml of 0.5% Triton X-100, 0.5% sodium deoxycholate, 20 mMTris-HCl pH 8. After loading the lysate, the column was washed with 40ml of the same solution and the elution of the bound material wascarried out by successive washings with buffers of increasing pH orionic strength: 1) 10 ml of 0.1% Triton X-100, 0.1M glycine pH 9; 2) 20ml of 0.1% Triton X-100, 0.1M glycine, pH 10; 3) 50 ml of 0.1% TritonX-100, 0.1M TEA, pH 11.5; and 4) 40 ml of 0.14M NaCl, 0.5% Triton X-100,0.01M Tris-HC1 pH 8. SDS-PAGE of the fractions showed that most of thebound material was eluted at pH 11.5.

The α-subunit of Mac-1 was isolated by preparative SDS-polyacryamide gelelectrophoresis of the affinity-purified antigen. After electroelution,the isolated α-subunit was precipitated with ethanol and reduced andalkylated. 50 μg of purified α-subunit was dissolved in 0.1M ammoniumbicarbonate, 0.1 mM calcium chloride, 0.3% zwittergent 3-14 and digestedwith 2% (w/W) trypsin at 37° C. for 6 hours, with further additions of1% tyrpsin every 2 hours. The resulting peptides were separated byreverse-phase HPLC on a C4 column (Vydac) and eluted with a gradient ofacetronitrile (0-60%) in 0.1% trifluoroacetic acid (TFA) for 2 hours.Collected fractions were concentrated and subjected to microsequencingon an applied Biosystems gas-liquid phase sequencer.

Immunoprecipitation with-subunit-specific monoclonal antibody showedthat Mac-1 was the prevalent molecule eluted from the anti-β monoclonalantibody-Sepharose column, as expected since neutrophils are the primarycell in the leukocyte lysate and express much more Mac-1 than LFA-1 orp150,95.

EXAMPLE 2 Isolation and Sequencing of cDNA Clones

The purified α-subunit was digested with trypsin and the resultingpeptides separated by reverse-phase HPLC and subjected to proteinmicro-sequencing (Table I). Comparison of the Mac-1 α-subunit peptidesequences with the sequence of the p150,95 α-subunit (Corbi, A. et al.,EMBO J. 6:4023-4028 (1987)) showed a high degree of similarity. Table Ishows the tryptic peptides of the Mac-1 alpha-subunit, and theirpositions in the cDNA-derived sequence. In the Table, positions withoutamino acid assignment are shown by "X;" uncertain positions are shown inparenetheses. Peptides were obtained from Mac-1 purified on IB4monoclonal antibody-Protein-A-Sepharose, except for peptide 86 that wasobtained from Mac-1 purified on LM2/1 monoclonal antibody-Sepharose, andpeptides 88 and 90 that were obtained from both sources. The peptidesequence used for the design of the oligonucleotide probe is underlined.

                                      TABLE I                                     __________________________________________________________________________    SEQUENCES OF TRYPTIC PEPTIDES OF Mac-1 ALPHA-subunit                          Peptide                                                                            Amino Acid Sequence       Residues                                       __________________________________________________________________________    14   T I Q N Q L R             307-313                                        32   V Q S L V L G A P R       400-409                                        43   Y V I G V G D A F R       267-276                                        44   Y Q H I G L V A M F R     410-420                                        53A  W Q C (D) A V L Y G E Q G Q P X G R                                                                     488-504                                        53B  E F V (S) X X (M) (E) (Q) (L)                                                                           155-164                                        54A  F (G) D P L G Y E D V I P E A D R                                                                       246-261                                        71   L F T A L F P F E K       744-753                                        79A  V D S D M N D A Y L G (Y) 379-390                                        79B  X Q (C) X I P F F G I Q E 1015-1026                                      86   G C P Q E D S D I A F L I D G S G S I I P H D F R                                                       127-151                                        88   T Q T V F F F P L D L S Y R                                                                             801-814                                        90   L X F S L V G T P L S A F G N L R P V L A E D A Q R                                                     718-743                                        __________________________________________________________________________

Peptide sequences were used to design 4 single sequence oligonucleotideprobes. The peptide 88 was selected to design a 42-mer oligonucleotidespecific for the α-subunit of Mac-1 because of its low level ofredundancy and its homology to a region of the p150,95 α-subunit closeto the C-terminus (i.e., towards the 3' end of a cDNA).

The oligonucleotides were end-labeled using T4 polynucleotide kinase andγ³² P!-ATP (Maniatis, T., et al. (In: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.(1982)) and used to screen a sized-selected cDNA library fromPMA-induced HL60 cells (Corbi, A. et al., EMBO J. 6:4023-4028 (1987)).5×10⁵ primary recombinants were plated, transferred to duplicatenitrocellulose filters, and prehybridized overnight as described (Corbi,A. et al., EMBO J. 6:4023-4028 (1987)). Hybridization with theoligonucleotides was done in 6×SSC, 0.1% SDS, 0.05% sodiumpyrophosphate, and 100 μg/ml of tRNA at 37° C. overnight. Filters werewashed in the same solution without the tRNA at room temperature for 30min. and at 45° C. for 15 minutes. Wet filters were exposed overnight topre-flashed X-Ray film with intensifying screen.

Phage plaques giving duplicate positive signals were obtained afterscreening with a 42-mer oligonucleotide:

    5'-ACCCAGGTGACCTTCTTCTTCCCCCTAGACCTGTCCTACCGG-3'

and were subjected to three additional rounds of subcloning andscreening with the same probe. Isolation of full-length cDNA clones wascarried out by re-screening the filters with an end-labeledoligonucleotide having the sequence:

    5'-GGATGGACTGGTAGACCTGACTGTAGGAGC-3'

and nick-translated probes from the 5' end of the partial cDNA cloneλM14.

The screening of the 5×10⁵ primary recombinants from the PMA-inducedHL-60 cDNA library with the 42-mer yield 16 positive clones and thelongest one (λM14, 2.9 kb) was selected for sequencing (FIG. 1). TheλM14 cDNA-derived amino acid sequence encodes four of the typticpeptides derived from the purified Mac-1 α-subunit. To isolate afull-length Mac-1 α cDNA clone, the library was re-screened with a 1.0kb EcoRI fragment and a 30-mer derived from the 5' end of λM14 andtwenty-four new cDNA clones extending towards the N-terminus of theprotein were selected. Isolation of the inserts of these 24 cDNA clonesshowed that three of them (λM23, λM42 and λM90) extend 2 kb 5' of λM14(FIG. 1).

λ23 is a full-length Mac-1 α cDNA clone. It encodes the proteinN-terminus (Miller, L. J. et al., J. Immunol. 138:2381-2383 (1987)) andthe tryptic peptides not detected in λM14. λM14 and λM23 exhibitidentical restriction maps in their overlapping regions.

EXAMPLE 3 Restriction Mapping and Sequencing

DNA from the positive phages was purified, cloned into pUC13, 18 or 19,and restriction mapped using standard procedures (Maniatis, T., et al.(In: Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y. (1982)). Restriction fragmentswere subcloned into M13 mp18 and mp19 and sequenced by the dideoxytermination method (Sanger, F. et al., Proc. Natl. Acad. Sci. (U.S.A.)74:5463-5467 (1977). Oligonucletide-primed DNA sequencing was used incases where no convenient restriction sites were available. The wholecoding region, the 5' untranslated region and more than 60% of the 3'untranslated region were sequenced in both strands. The 3' untranslatedregions of the cDNA clones λM23 and λM90 were subjected to dideoxysequencying of plasmid DNA and using the Erase-a-base system (Promega)to make deletions according to the manufacturer's instructions(Henikoff, S. Gene 28:351-359 (1984)).

The composite cDNA sequence of λM23 contains 4740 bp and has a long openreading frame of 3534 nucleotides, leaving a 5' untranslated region of72 bp and a 3' untranslated region of 1.2 kb (FIG. 2). The 3'untranslated region of λM14 contains an inverted stretch of poly-(CA)(nucleotides 3667-3862) (Sun, L. et al., Nucl. Acid Res. 12:2669-2690(1984)), a partial Kpn I interspersed repeat (nucleotides 4566-4631)(Sun, L. et al., Nucl. Acid Res. 12:2669-2690 (1984)), and ends with astretch of more than 40 adenosines. There are 2 consensuspolyadenylation signals at nucleotides 4191 and 4678. Analysis of the 3'untranslated regions from λM14, λM23, and λM90 indicates that the firstpolyadenylation signal is used in λM23 and the second one in λM14 andλM90, indicating that both polyadenylation signals are functional.Restriction mapping of 15 additional cDNA clones suggests that bothpolyadenylation sites are used with equal frequency. The 3' untranslatedregion of λM90 (and λM42) lack 440 bp found between nucleotides 3629 and4070 in λM14 and λM23 (FIG. 1). The sequences GAA/GTATCC and AAG/A atthe boundaries of the deletion (arrows, FIG. 2) conform to the GT/AGrule for splicing sites (Mount, S. M., Nucl. Acid Res. 10:459-472(1982)) and thus the two different classes of cDNA's appear tocorrespond to alternatively spliced mRNA's.

The open reading frame from λM14/λM23 translates into a protein of 1137residues, with a signal peptide of 16 amino acids defined by thepreviously reported Mac-1 α-subunit N-terminal sequence (Miller, L. J.et al., J. Immunol. 138:2381-2383 (1987); Pierce, M. W. et al., Biochim.Biophys. Acta 874:368-372) (FIG. 2). In addition to the agreement withthe protein N-terminal sequence, the 186 residues determined by trypticpeptide sequencing (Table I) agreed perfectly with the translatedsequence. This confirmed the isolation of authentic Mac-1 α-subunit cDNAclones. The amino acid sequence of the Mac-1 α-subunit has thecharacteristics of a classical transmembrane protein, with an N-terminal1092-residue domain, a 25-residue hydrophobic putative transmembranedomain, and a 19 residue C-terminal hydrophilic domain (FIG. 2). Thepresence in the N-terminal 1092-residue domain of 19 potentialN-glycosylation sites (Asn-Xaa-Ser/Thr), one of which was sequenced inpeptide 90 (residues 718-743), confirms that this is the extracellulardomain. The predicted molecular weight of the protein is 125,611daltons, consistent with previous estimations after N-glycanasetreatment of the α-subunit of Mac-1 (137,000 Mr) (Miller, L. J. et al.,J. Immunol. 139:842-847 (1987)). Assuming 2,500 Mr per high mannosecarbohydrate, Mr=173,001 is predicted for the Mac-1 α-subunit precursor,compared to the observed M=160,000 (Miller, L. J. et al., J. Immunol.139:842-847 (1987)). After carbohydrate processing, the Mac-1 α-subunitis 170,000 Mr.

The primary structure of the α-subunit of Mac-1 suggests the presence ofseven internal repeats (FIG. 3). Repeats V, VI, and VII show the highestdegree of similarity to one another which is statistically significant(p<10⁻² to p<10⁻⁴) and contain sequences similar to the divalentcation-binding EF-hand loop motif of proteins like calmodulin andparvalbumin (Szebenyi, D. M. E. et al., Nature 294:327-332 (1981)) (FIG.3), correlating with the divalent cation requirements of theMac-1-mediated adhesion (Wright, S. D. et al., Proc. Natl. Acad. Sci.(U.S.A.) 80:5699-5703 (1983)). Repeats I-IV lack the EF-hand loop-likesequences but contain the highly conserved sequences Y F G A S/A L and LV T V G A P flanking the center of the repeats (FIG. 3). These consensusflanking sequences are also conserved in other integrins (FIG. 3). Thepresence of the seven repeats suggest that much of the N-terminalportion of the Mac-1 α-subunit may have arisen by duplication events.

EXAMPLE 4 Northern Blot Analysis

Adherent mononuclear cells from peripheral blood were isolated byFicoll-Hypaque centrifugation and incubation of the mononuclear cells intissue culture plates with RPMI 1640 and 10% fetal calf serum for 30min. at 37° C. Non-adherent cells were removed from the plates byextensive washing with RPMI 1640. Adherent cells were detached from theplates by incubation with 10 ml of PBS, 5 mM EDTA, for 15 minutes at 37°C. More than 95% of the adherent cells were positive for the presence ofMac-1 as detected by indirect immunofluorescence. PMA-treatment of thecell lines HL-60 and U937 was performed as described (Miller, L. J. etal., J. Immunol. 137:2891-2900 (1986)). total RNA was extracted fromperipheral blood adherent cells, as well as from the cell lines SKW3,JY, HL-60, and U937, using guanidine isothiocyanate (Maniatis, T., etal. (In: Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y. (1982)). 10 or 20 μg from eachsample was run on a 1% formaldehyde-agarose gel and transferred onto anylon membrane. Hybridizations were performed using the 2.3 kb Eco RIfragment from λM23 as a probe.

The cell surface expression of the Mac-1 α/β complex was found to bealmost exclusively restricted to cells of the myeloid lineage (Miller,L. J. et al., J. Immunol. 137:2891-2900 (1986)). Northern blots showedthat the Mac-1 α-subunit mRNA is 4.7 kb and was present in monocytes andmyeloid cell lines, but not in T or B cell lines.

Mac-1 expression was regulated during leukocyte differentiation, andcould be induced in myelomonoctic cell lines by culture with phorbolester for 1-3 days (Miller, L. J. et al., J. Immunol. 137:2891-2900(1986)). Northern blot analysis revealed that the steady-state level ofMac-1 α RNA in the HL-60 and U937 mylelomonocytic cell lines wasextremely low or nil (Miller, L. J. et al., J. Immunol. 137:2891-2900(1986)). PMA-treatment of both cell lines induced expression of theMac-1 α-subunit mRNA and increased the expression of the β-subunit mRNA.These findings are concordant with previous studies on the biosynthesisof the Mac-1 α and β-subunits (Miller, L. J. et al., J. Immunol.139:842-847 (1987)), on the surface expression of the Mac-1 α/β complexon these cell lines (Miller, L. J. et al., J. Immunol. 137:2891-2900(1986)), and suggest that the cell surface expression of Mac-1 isregulated by mRNA level. Similarly, treatment of the murinepremyelocytic cell line M1 with γ-interferon induces expression of themuring Mac-1 α-subunit mRNA (Sastre, L. et al., Proc. Natl. Acad. Sci.(U.S.A.) 83:5644-5648 (1986)). Two Mac-1 α-subunit messages ofapproximately 4.7 and 4.4 kb were resolved in the myelomonocytic celllines HL60 and U937 upon prolonged electrophoresis of the RNA. Thepresence of multiple Mac-1 α mRNA species may be the result of thealternative use of the two polyadenylation signals or alternativesplicing. The size of the mRNA species is consistent with thesepossibilities. Southern blot analysis under stringent conditions onhuman DNA demonstrated that Mac-1 α-subunit is encoded by a single copygene.

EXAMPLE 5 Sequence Homologies

The sequence of Mac-1 α-subunit was compared with the protein sequencedatabase in the National Biomedical Research Foundation (NBRF)(Washington, D.C.). The ALIGN program was used for alignment ofsequences (Dayhoff, M. O. et al., Met. Enzymol. 91:524-545 (1983)). Thestatistical significant of the alignments was assessed obtaining thealignment scores for 100 random permutations of the aligned sequencesand calculating the number of standard deviations between the mean ofthe scores for the randomized comparisons and the score of the actualalignment. The scoring used the 250 PAM mutation data matrix, with a gappenalty=6 and a bias=6.

EXAMPLE 6 Mac-1 as a Member of the Integrin Gene Superfamily

The determination of the primary structure of the Mac-1/LFA-1/p150,95common β-subunit (Kishimoto, T. K. et al., Cell 48:681-690 (1987); Law,S. K. A. et al., EMBO J. 6:915-919 (1987)) and the α-subunit of p150,95(Corbi, A. et al., EMBO J. 6:4023-4028 (1987)) has shown that theleukocyte adhesion receptors are evolutionary related to theextracellular matrix (ECM) receptors, and led to the concept of a genesuperfamily of cell-cell and cell-matrix receptors termed "integrins"(Hynes, R. O. Cell 48:549-554 (1987)).

Three subfamilies of integrin molecules, each with a distinct β-subunit,have been defined, namely the fibronectin receptor subfamily (sharingintegrin β₁), the leukocyte adhesion receptor subfamily (sharingintegrin β₂), and the vitronectin receptor-IIb/IIIa subfamily (sharingintegrin β₃).

Comparisons of the amino acid and nucleic acid sequences which encodethe integrins with those of the Mac-1 α-subunit were undertaken todefine the relationships among the α-subunits of the different integrinsubfamilies (FIG. 4). The α-subunits of Mac-1 and p150,95 were found tobe 63% identical at the amino acid level and 68% identical at thenucleotide level. The high degree of structural similarity between theα-subunits of Mac-1 and p150,95 is reflected at the functional level:Mac-1 and p150,95 exhibit iC3b-binding ability (Beller, D. I. et al., J.Exper. Med. 156:1000-1009 (1982); Micklem, K. J. et al., Biochem. J.231:233-236 (1985)) and both proteins are known to play a role inneutrophil aggregation and neutrophil and monocyte adhesion toendothelials cells (Anderson, D. C. et al., J. Immunol. 137:15-27(1986); Vedder, N. B. et al., J. Clin. Invest. 81:672-682 (1988); TeVelde, A. A. et al., Immunol. 61:261-267 (1987)). The α-subunit of LFA-1is 35% identical to the α-subunits of Mac-1 and p150,95. The α-subunitsof the fibronectin receptor, vitronectin receptor and the glycoproteinIIb are 40% identical to one another. Since the α-subunits of Mac-1 andp150,95 are 25% identical to the α-subunits of the three ECM receptors,the α-subunits of Mac-1, P150,95, and LFA-1 are more closely related toeach other than to the rest of the integrin α-subunits. The leukocyteα-subunits also resemble one another in containing a segment of 187residues not found in the ECM α-subunits (amino acids 150-338 in theα-subunit of Mac-1), and in lacking a region of 28 amino acids (gap atresidue 1002 in Mac-1) where the ECM receptor α-subunits areproteolytically cleaved during processing to generate twodisulfide-linked chains (Ruoslahti, E. et al., Science 238:491-497(1987)).

The area of highest extended identity between Mac-1 α-συβυνιτ and therest of the integrin α-subunits lies between residues 434-592, preciselythe boundaries of the three internal repeats containing putativedivalent cation-binding sequences. Over this region, Mac-1 α-subunitshows 88% identity to p150,95 α-subunit at the amino acid level and 90%at the nucleotide level, and the percentage of identity to the ECMreceptor integrin α-subunits is 38%. The ECM receptor α-subunits havefour putative divalent cation-binding sites within their primarystructure (Suzuki, S. et al., J. Biol. Chem. 262:14080-14085 (1987);Argraves, W. S. et al., J. Cell Biol. 105:1183-1190 (1987); Poncz, M. etal., J. Biol. Chem. 262:8476-8482 (1987)), which align with the repeatsIV-VII in the α-subunits of Mac-1 and p150,95; repeat IV in Mac-1 andp150,95 does not contain the putative divalent cation-binding sequence(FIG. 3). Ligand-binding by the ECM receptor integrins iscalcium-dependent (Ruoslahti, E. et al., Science 238:491-497 (1987)) andin the case of the α-subunit of the glycoprotein IIb/IIIa the binding ofradioactive calcium has been verified (Ruoslahti, E. et al., Science238:491-497 (1987)). The high degree of conservation of these regionssuggests a role in maintaining receptor conformation or a directinvolvement in ligand binding.

An additional region of high conservation among the integrin α-subunitsare the membrane spanning regions. The transmembrane domain of Mac-1 αexhibits 88% identity with the one in p150,95 α and 40-50% identity withthose of the IIb, VNR and FNR α-subunits (Tamkun, J. W. et al., Cell46:271-282 (1986); Argraves, W. S. et al., J. Cell Biol. 105:1183-1190(1987); Poncz, M. et al., J. Biol. Chem. 262:8476-8482 (1987)) (FIG. 4).A similar conservation has been noted in the transmembrane andcytoplasmic domains of the different β-subunits (Kishimoto, T. K. etal., Cell 48:681-690 (1987); Law, S. K. A. et al., EMBO J. 6:915-919(1987)); Tamkun, J. W. et al., Cell 46:271-282 (1986); Fitzgerald, L. A.et al., J. Biol. Chem. 262:3936-3939 (1987); Argraves, W. S. et al., J.Cell Biol. 105:1183-1190 (1987)). Based on this high degree ofconservation it is conceivable that these regions may play a role in theregulation of the ligand-binding (Vedder, N. B. et al., J. Clin. Invest.81:672-682 (1988)) through interactions with other membrane componentsor with the cytoskeleton. The finding that the fibronectin receptorbinds talin (Horwitz, A. et al., Nature 320:531-533 (1986)), that LFA-1mediated interactions are dependent on the integrity of the cytoskeleton(Springer, T. A. et al., Ann. Rev. Immunol. 5:223-252 (1987)), and thatLFA-1 co-caps with talin after phorbol ester stimulation (Burn, P. etal., Proc. Natl. Acad. Sci. (U.S.A.) 85:497-501 (1988)) further supportsthe involvement of these domains in cytoskeletal interactions and insignal tranduction.

EXAMPLE 7 Mapping of the Mac-1 Alpha Gene

The Mac-1, LFA-1, and p150,95 α and β-subunit genes have been located bySouthern blot on mouse x human somatic cell hybrids and by chromosomalin situ hybridization using cDNA probes. The genes encoding theα-subunits of LFA-1, Mac-1 and p150,95 map to chromosome 16, betweenbands p11-p13.1, defining a gene cluster involved in leukocyte adhesion.The common structural characteristics and the close proximity of thethree α-subunit genes strongly suggest that the genes for the α-subunitsof Mac-1, p150,95, and LFA-1 evolved by gene duplication events, andthat these gene duplications took place after the divergence of thedifferent integrin α-subunit subfamilies.

EXAMPLE 8 Homology with von Willebrand factor and factor B

As previously described for p150,95 (Corbi, A. et al., EMBO J.6:4023-4028 (1987)), there is a region of 187 amino acids in the Mac-1α-subunit (resides 150-338) without counterpart in the 3 sequenced ECMreceptor integrin α-subunits. This region is referred to as the Ldomain, because of its presence in leukocyte integrins. The abovediscussed search of the National Biomedical Research Foundation sequencedata base using the FASTP program revealed homology of residues 128-314of the Mac-1 α-subunit with von Willebrand factor (vWF) (Shelton-Inloes,B. B. et al., Biochem. 25:3164-3171 (1986)) (FIG. 5). The homologousregions are of particular interest because they correspond in Mac-1almost exactly to the L domain, and in vWF to the type A domain, adomain previously defined by its presence in 3 homologous tandem repeatsof 200 residues denoted A1 (497-716), A2 (717-909), and (910-1111)(Shelton-Inloes, B. B. et al., Biochem. 25:3164-3171 (1986)). Thep150,95 α-subunit (FIG. 4) and the murine Mac-1 α-subunit also showhomology to the vWF A domains. Since the vWF A domains have previouslyhave shown to be homologous to factor B (Shelton-Inloes, B. B. et al.,Biochem. 25:3164-3171 (1986); Mole, J. E. et al., J. Biol. Chem.259:3407-3412 (1984)), a component of the alternative complement pathwaywhich interacts with C3, homologies with factor B and with its homologuein the classical pathway, C2 (Bentley, D. R., Biochem. J. 239:339-345(1986)) were also examined (FIG. 5). The homologous domain in factor Bis clearly demarcated on the N-terminal side by the site at which factorB is cleaved to give the active Bb factor, and on the C-terminal side bythe serine protease domain (Mole, J. E. et al., J. Biol. Chem.259:3407-3412 (1984)); Bentley, D. R., Biochem. J. 239:339-345 (1986))(FIG. 6). Evaluation with the ALIGN program (Dayhoff, M. O. et al., Met.Enzymol. 91:524-545 (1983)) showed that the homology of Mac-1 with vWFdomains A1, A2, and A3 and with factor B is highly statisticallysignificant (Table II), and, thus, that the homologous amino acidsegments in each of these proteins must have evolved from a singleprimordial domain. Although the homology of Mac-1 with C2 is notstatistically significant, factor B shows significant homology to C2 andserves as an evolutionary link, showing that the segment in C2 evolvedfrom the same primordial domain. Table II compares the alignment scoresof the Mac-1 alpha-subunit leukocyte specific domain (128-314), with theA repeats of von Willebrand factor (A1:509-692; A2:730-903;A3:923-1103), Factor B (240-443), and C2 (228-434). The table wascompiled using the ALIGN program. The alignment scores are presented instandard deviation units. The probability of the alignments (shown inFIG. 6) have occurred by chance is shown in parentheses below eachalignment score.

                  TABLE II                                                        ______________________________________                                        SIGNIFICANCE OF THE ALIGNMENT SCORES OF THE Mac-1                             ALPHA-subunit LEUKOCYTE-SPECIFIC DOMAIN WITH THE von                          WILLEBRAND FACTOR A REPEATS, FACTOR B AND C2                                  vWF A1      vWF A2   vWF A3   Factor B                                                                             C2                                       ______________________________________                                        Mac-1  7.4       7.2     8.1    6.9    1.8                                           (<10.sup.-11)                                                                          (<10.sup.-11)                                                                          (<10.sup.-15)                                                                        (<10.sup.-10)                                                                        (<10.sup.-1)                           vWf A1 --       10.3     9.1    7.0    4.6                                                    (<10.sup.-23)                                                                          (<10.sup.-18)                                                                        (<10.sup.-11)                                                                        (<10.sup.-5)                           vWf A2 --       --       13.4   4.0    4.1                                                             (<10.sup.-23)                                                                        (-10.sup.-4).sup.                                                                    (<10.sup.-4)                           vWf A3 --       --       --     4.8    3.2                                                                    (<10.sup.-5).sup.                                                                    (<10.sup.-2)                           Factor B                                                                             --       --       --     --     23.8                                                                          .sup. (<10.sup.-23)                    ______________________________________                                    

EXAMPLE 9 The Ligand Binding Site of the Mac-1 Alpha-subunit

The homology between factor B and the L domain, and the ability offactor B to bind C3b, support the conclusion that the L domain is theiC3b ligand-binding site of Mac-1 and p150,95. It is also of interestthat binding of Bb to C3b requires Mg⁺² (Muller-Eberhard, H. J. et al.,Adv. Immunol. 29:1-53 (1980)). Similarly, binding of isolated Mac-1 andp150,95 to iC3b-sensitized cells or to iC3b-Sepharose requires divalentcations (Wright, S. D. et al., Proc. Natl. Acad. Sci. (U.S.A.)80:5699-5703 (1983); Micklem, K. J. et al., Biochem. J. 231:233-236(1985)). A putative divalent cation binding site in the L domain may berepresented by one sequence motif containing dG and another containtingDG and GD which are concerned in Mac-1, p150,95, and factor B(underlined in FIG. 5). If these sites were contiguous in the 3dimensional structure they could form a divalent cation binding sitesimilar in sequence to those present in internal repeats V to VII of theα-subunit of Mac-1. The L domain and the following region containingrepeats IV-VII are relatvely free of N-linked glycosylation sites andcysteines (FIG. 6), allowing them to be accessible and conformationallyflexible. The idea that the L domain could be involved in recognition ofiC3b raises the possibility of recognition of a sequence in iC3bdistinct from RGD. Although RGD is present in iC3b, evidence that it isimportant in recognition by Mac-1 (Wright, S. D. et al., Proc. Natl.Acad. Sci. (U.S.A.) 84:1965-1968 (1987)) remains to be confirmed bypeptide inhibition data.

Both the A1 and A3 domains of vWF bind collagen (Girma, J. P. et al.,Blood 70:605-611 (1987)) and the A1 domain binds the plateletglycoprotein Ib and heparin (Girma, J. P. et al., Blood 70:605-611(1987)). The alternative complement pathway in which factor B plays animportant role is the most primitive mechanism of the immune system fordistinguishing self from non-self (Muller-Eberhard, H. J. et al., Adv.Immunol. 29:1-53 (1980)). Thus, the 200 amino acid domain appears to bea primitive recognition unit which has been duplicated and embedded in anumber of proteins which have evolved to play diverse recognitionfunctions in hemostasis (vWF), the extracellular matrix (CMP) complementactivation (factor B and C2), and complement receptor and cell-cellinteractions (leukocyte integrins).

EXAMPLE 10 The Phylogenetic Relationship Between the Integrins

The ECM receptor integrins are phylogenetically ancient as shown at thelevel of sequence homology in Drosophila (Ruoslahti, E. et al., Science238:491-497 (1987)) and by immunological cross-reaction with proteins ofsimilar size in nematodes and fungi. These results demonstrate that theMac-1 α-subunit evolved by the introduction of a primordial recognitiondomain into the ECM receptor-type of α-subunit evolved by theintroduction of a primordial recognition domain into the ECMreceptor-type of α-subunit (FIG. 6). The introduction of the extradomain may have increased the potential for recognition of diverseligands by the leukocyte integrins and may explain their somewhatdifferent ligand specificity, since recognition of the ICAM-1 ligand byLFA-1 does not involve RGD (Marlin, S. et al., Cell 51:813-819 (1987);Staunton, D. E. et al., Cell 52:925-933 (1988); Simmons, D. et al.,Nature 331:624-627 (1988), all of which references are incorporatedherein by reference).

The availability of cDNA clones for both the α and β-subunits of Mac-1allows the identification of the distinct ligand binding sites involvedin iC3b binding and in cell-cell adhesion, and the testing of thehypothesis that cell stimulation results in a conformational change inthe ligand binding site, transmitted from the cytoplasmic or membranedomains, that alters affinity for ligand.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

What is claimed is:
 1. An isolated nucleic acid molecule comprising thenucleic acid sequence depicted in FIG.
 2. 2. An isolated nucleic acidmolecule which encodes the amino acid sequence of human MAC-1alpha-subunit depicted in FIG.
 2. 3. The isolated nucleic acid moleculeof claim 2, wherein said nucleic acid molecule is contained in a vector.4. The vector of claim 3, wherein said vector is an expression vector.